Long-term goals of the proposed research are development of simple, quantitative measures of oral microbial (saliva and plaque biofilm) community composition and physiology, examination of temporal and spatial variations in structure/function relationships in oral communities, and characterization of nutrient- and environment-dependent shifts in community composition and physiology. These goals seek to elucidate the shifts natural to the progression of plaque biofilms, especially pathogen communities of periodontal disease. The present proposal seeks to define a species-composition measurement using phospholipid-bound fatty-acid (PLFA) profiles of whole-community biomass, and will validate that measurement by correlation with checkerboard hybridization and cultivable flora methods. Analysis of biofilm architecture will be performed using electron microscopy and laser confocal microscopy, and correlation of that data with compositional and physiological data will be performed. Community physiology measures will be established and applied to in vitro biofilm microcosms and defined-species consortia. "Functional blocks" will be defined by characterizing enzymatic activities (proteases, mucin-specific exoglycosidases, urease) as indicators of nutrient-protein catabolism, mucin catabolism, and urea hydrolysis; the rate of glucose fermentation and of acid production as indicators of saccharolytic fermentation and ammonia release as an indicator of amino acid metabolism. Whole-community metabolic profiles will be performed using BiologTM and API-ZymTM technologies. The effects of shifts in nutrient composition and environmental pH will be examined by monitoring community physiology, community composition, and architecture. Hypotheses for the proposed work are: 1) that application of PLFA analysis principles proven effective in community taxonomy of natural samples to oral biofilms will yield rapid and significant information on community composition. PLFA analysis will permit simple, frequent, and information-rich patient monitoring and thus improve our understanding of microbial community-composition shifts that underlie the progression of periodontal disease and dental caries. 2) that bacterial community composition and community physiology respond in a understandable manner to shifts in nutrient composition and environmental pH. Composition and physiology are related though not necessarily directly linked. Ecologically induced shifts are presumed to be key factors in the progression of periodontal disease; knowledge of the controls over normal and pathogen communities in plaque will enhance our understanding of cause-and-effect in periodontal disease and caries as well as underpinning development of new intervention strategies.